Native covalently closed circular DNA's are found in the supercoiled or superhelical state. An understanding of the development of this structure and the relationship between supercoiling and secondary structure are not clear at the present moment. This is indicated by our recent discovery of the altered secondary structure in two viral supercoiled molecules. The continued investigation of PM2 and 0X-RF DNA's by chemical probes strongly supports our original observations of the altered secondary structure as well as a new dynamic model of the behavior of these molecules. This data coupled with recent biological studies in other laboratories strongly suggest that the supercoiled state may represent a biologically organized structure that serves important functions. The objectives of this study are: 1. A full understanding of the secondary structure in the altered regions; 2. The development of suitable experiments to test the effect of the secondary structure on various aspects of biological function. A wide range of experiments are planned to meet our objectives. These include: a. The continuation of structural studies of supercoiled DNA using reaction kinetics, solvent effects and chemical probes; b. Chemically modified DNA will be used to reveal structure-function relationships by studying in vitro RNA polymerase binding and transcription; c. These modified molecules will also serve to reveal the distribution and genetic location of the altered structure by suitable use of restriction enzymes and genetic analysis; d. In vivo functions will be tested using chemically modified DNA with different degrees of bound reagent; e. The knowledge gained above will serve readily in a study of supercoiled-replication intermediates. This comprehensive research proposal should allow us to develop a great understanding of structure-function relationships of supercoiled DNA.